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Biomedical subjects

T Shinohara

Publications and source records attributed to T Shinohara.

At least 325 records · Page 18Linked to original sources
PubMed1986

Sequence analysis of bovine retinal S-antigen. Relationships with alpha-transducin and G-proteins.

S-Antigen is a major soluble protein of the retina and pineal. It is capable of inducing experimental autoimmune uveitis (EAU) in laboratory animals and also seems to play an important role in the visual cycle. The results of partial cDNA sequence analysis reveal interesting homologies with alpha-transducin, a GTP-binding protein of retina and other purine nucleotide-binding proteins. In particular S-antigen shows over 50% identity to the proposed pertussis toxin ADP-ribosylation site of alpha-transducin. It also contains the Gly-X-X-X-X-Gly-Lys pattern common to phosphoryl binding sites. A possible relationship between S-antigen and purine nucleotide-binding proteins is discussed. There is also evidence for a repetitious beta-structure in the C-terminal half of S-antigen, with a monoclonal antibody epitope in a helical region at the C-terminus.

Amino Acid Sequence↗
PubMed1986

S-antigen: identification of the MAbA9-C6 monoclonal antibody binding site and the uveitopathogenic sites.

The location of the monoclonal antibody, MAbA9-C6, binding site and two uveitopathogenic sites in S-antigen have been determined. Using cyanogen bromide, S-antigen was cleaved into nine peptides, designated C1 to C9. MAbA9-C6 bound selectively to one large peptide designated C5, consisting of 122 amino acids. Six peptides (20 to 22 amino acids in length) designated 2,3,K,L,N and M, corresponding to the entire length of peptide C5, were synthesized chemically. In a radioimmunoassay and a dot-binding immunoassay, MAbA9-C6 bound selectively to one of the six peptides, peptide 3, indicating that this region of peptide C5 contains the MAbA9-C6 binding site. Twelve smaller peptides (ten amino acids in length), corresponding to the amino acid sequence of peptide 3, were synthesized and used in a competitive inhibition binding assay. These studies localized the MAbA9-C6 binding site to a small region within peptide 3. In addition, peptide K and peptide M were highly pathogenic for the induction of experimental auto-immune uveitis (EAU). Clinical and histological evidence of a severe uveo-retinitis, indistinguishable from that seen with native S-antigen, was documented in Lewis rats immunized with the synthetic peptides (50 micrograms), 11-12 days following immunization. Our results show that the MAbA9-C6 binding site and the two uveitopathogenic sites lie in close proximity to each other within the region of S-antigen corresponding to peptide C5. Furthermore, microcomputer analysis of the average hydrophilicity/hydrophobicity values of the amino acid sequence corresponding to peptide C5 shows that the MAbA9-C6 binding site and one uveitopathogenic site (peptide K) lie on the adjacent peaks. The significance of these findings and their relationship to the role of S-antigen in the pathogenesis of EAU and the phototransduction of vision is discussed.

Amino Acid Sequence↗
PubMed1985

Selective loss of a family of gene transcripts in a hereditary murine cataract.

The eye lens of the Fraser mouse contains a dominantly inherited cataract with reduced amounts of seven distinct but homologous gamma crystallins encoded by a family of gamma-crystallin genes. The results of experiments with cultured lenses, cell-free RNA translation, and Northern blot hybridization indicated a specific loss of the family of gamma-crystallin messenger RNA's in the Fraser mouse lens. Southern blot hybridization of genomic DNA's from normal and Fraser mice showed no differences in gamma-crystallin coding sequences.

Animals↗
PubMed1985

Differential synthesis of rat lens proteins during development.

Developmental regulation of crystallin protein synthesis was observed in rat lenses between embryonic day 19 and postnatal day 21. Studies on lenses incubated in [35S]-methionine and on lens messenger RNAs translated in a reticulocyte lysate showed that several new polypeptides were synthesized in the lens beginning approximately 1 week after birth. One new polypeptide which had a molecular weight of 27 000 comigrated with the beta crystallins on SDS-PAGE and became a predominant component in older lenses. By crossed rocket immunoelectrophoresis and isoelectric focusing, synthesis of several native beta crystallins and one gamma crystallin was detected only in the postnatal lens. Many crystallin proteins were synthesized in the embryonic and the postnatal lens and did not change during the time period studied. These data suggest a differential regulation of the crystallin proteins during development. It appears that the lens undergoes a transition from embryonic to adult crystallin expression during the first weeks after birth. Factors such as maturation of the retina may be necessary for this transition.

Aging↗
PubMed1984

Analysis of the mouse gamma-crystallin gene family: assignment of multiple cDNAs to discrete genomic sequences and characterization of a representative gene.

Blot hybridization analysis of mouse DNA with gamma-crystallin-specific cDNAs has detected the presence of a multigene family comprised of at least four related genes. The detailed structure of one of these genes, mouse gamma 4-crystallin (M gamma 4.1), and its corresponding cDNA has been determined. The gene spans approximately 2.6 kilobases (kb) and contains two introns. The gene predicts a polypeptide of 174 amino acids that shares extensive sequence homology with gamma-crystallin polypeptides of other species. The two similar structural domains of the protein correspond exactly to the second and third exons of the gene, supporting an exon-duplication model of gene evolution. The similarity in structure of this gene to that recently reported for a gamma-crystallin gene of the rat (1) suggests that a common structure may exist for all gamma-crystallin genes of the two species. Moreover, a highly conserved region, 50 nucleotides in length, immediately precedes the TATA box of both the mouse and rat genes, suggesting that this sequence may be important in gene regulation.

Amino Acid Sequence↗
PubMed1984

GENAS: a database system for nucleic acid sequence analysis.

A database system, named GENAS (GENe Analyzing System), for computer analysis of sequence was constructed using Adbis which is a relational database management system (1). GENAS enables us to retrieve any sequence data from EMBL nucleotide sequence data library (2) and readily to analyze them (if necessary, together with private data) by various application programs in a interactive manner. Analysis of structure of replication origin of replicons was demonstrated using this system.

Base Sequence↗
PubMed1984

Crystallin genes: templates for lens transparency.

Analysis of recombinant DNAs provides new information on the basis of crystallin evolution and diversity. All crystallin genes contain introns. Two similar, tandemly linked chicken delta-crystallin genes, which probably arose by gene duplication, contain at least 16-17 introns. In the beta-crystallins three introns are situated between exons encoding the structural motifs of the protein, thus relating gene and protein structure. The structurally similar beta- and gamma-crystallins are coded by separate gene families which apparently arose by successive duplications of a common ancestral gene. The N-termini (5' end of gene) of the beta-crystallins appear to have diverged, while the 3' ends have been conserved. In the single murine alpha A-crystallin gene, coding information (the insert exon) for the alpha Ains peptide is contained within an intron. Alternative RNA splicing of this gene gives both the alpha A2 and the alpha Ains crystallin mRNAs. Thus, molecular genetics is providing a deeper appreciation of evolutionary events and is serving to redefine the crystallins in terms of their genes. Since the crystallins are so abundant in the lens, greater understanding of their polypeptide and gene structure should contribute to our understanding of and ability to treat cataract.

Amino Acid Sequence↗
PubMed1984

Effects of alcohols on ADP-induced aggregation and membrane fluidity of gel-filtered bovine blood platelets.

The effects of four alcohols--n-propyl, n-butyl, n-amyl and n-hexyl alcohol--on the ADP-induced aggregation of gel-filtered bovine platelets were examined. All four alcohols inhibited the aggregation, the order of their effects being n-propyl less than n-butyl less than n-amyl less than n-hexyl. Comparison of the inhibitory effects of the alcohols with their physico-chemical properties showed that their degrees of inhibition depended on their hydrophobicities. Moreover, it was suggested that their interaction with the lipid layer of the membrane was important for the inhibition. Studies on the effects of alcohols on the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene-labeled platelets showed that the membrane fluidity of the platelets increased in the same concentration range in which aggregation inhibition was observed. Since the alcohols inhibited aggregation without affecting Ca2+ mobilization in the platelets, as revealed in this study, it was concluded that inhibition of platelet aggregation was due to perturbation of membrane lipids by the alcohols. This hypothesis is supported by several recent studies on the effects of cholesterol and cations, which suggest that a relatively rigid membrane favors platelet aggregation.

1-Butanol↗
PubMed1984

Defective terminal maturation along monocyte-macrophage lineage in chronic myelomonocytic leukemia.

The maturation process of leukemic myelomonocytoid cells derived from peripheral blood of two typical cases of chronic myelomonocytic leukemia was investigated in vitro. In both cases, liquid culture of the leukemic cells in the presence or absence of 12-o-tetradecanoyl phorbol-13-acetate yielded either monocyte-macrophages or mature granulocytes, respectively, when examined by May-Giemsa staining. However, using cytochemical criteria, both of these mature cell fractions were found to be heterogenous and still contained substantial numbers of cells positive for both nonspecific and chloroacetate esterases. Phagocytic activity was markedly defective in the monocyte-macrophages recovered from culture as well as in freshly-isolated peripheral monocytes in both patients. In addition, active oxygen production from isolated monocytes stimulated by zymosan was also decreased in one of the cases. The results indicate that at least in some cases of chronic myelomonocytic leukemia, leukemic cells have the potential to differentiate in vitro to monocyte-macrophages which, however, show only limited development of functions, as well as freshly-isolated monocytes from peripheral blood.

Aged↗
PubMed1984

Hemin-induced conversion of pyruvate kinase isozymes in K562 cells.

The effects of hemin on the conversion of pyruvate kinase (PK) isozymes from the M2-type to the L-type in K562 cells were investigated. Immunofluorescence, ion exchange chromatography, and electrophoretic studies showed that the untreated K562 cells contained only the M2-type PK, while eight to 20 days after induction with hemin, concomitant with hemoglobin F synthesis, L-type PK levels increased while M2-type PK levels decreased. Electrophoretic study revealed three hybrid isozymes of the L-type and M2-type PK. We conclude that the conversion of PK isozymes from the M2-type to the L-type in erythroid precursor cells occurs in the early stage of maturation.

Cell Line↗
PubMed1983

Schiff's base formation in the lens protein gamma-crystallin.

Uniquely among the soluble lens-specific proteins, gamma-crystallin is capable of binding the strongly chromophoric aldehyde retinal. A role for gamma-crystallin in protecting lens components from toxic aldehydes resulting from membrane oxidation is proposed and a molecular model of the probable interaction site is presented. The sequence of a tetrapeptide at this site is identical to that of the retinal binding site of bacteriorhodopsin.

Animals↗